The invention relates to a method for the selection of human cells for the preparation of human proteins by endogenous gene activation in order to produce human proteins in economical yields and in a form which is suitable for the production of a pharmaceutical preparation. The invention furthermore relates to a method for the production of human proteins in a cell line identified in this manner.
The production of human proteins by endogenous gene activation in a human cell line is known. For example, WO93/09222, WO94/12650, and WO95/31560 describe the production of human erythropoietin and other human proteins in human cell lines by endogenous gene activation.
In the documents cited, however, there is no mention of the fact that certain criteria must be observed in selecting the cell lines used for the production of human proteins. Therefore there can be no assurance that the desired human protein can be obtained in the desired yield and form, and free of contamination in the cell line chosen for its production. Accordingly, in the above-named documents only generally low yields of human proteins are achieved.
It was the objective of the present invention to eliminate the disadvantages of the state of the art and especially to offer criteria for the selection of human starting cell lines which are suitable for an endogenous activation of a predetermined target gene.
This objective is attained by a method for the selection of human cell lines for the production of human proteins by the activation of a target gene endogenously present in the cell line, characterized in that
(a) a human cell line is tested for the presence of the following features:
(i) a target gene with the desired nucleic acid sequence,
(ii) at least 5 population doublings within 14 days in a suspension culture, and
(iii) at least 5 population doublings within 14 days in a serum-free culture medium, and
(b) using a cell line having features (i), (ii), and (iii) as starting cell line for the endogenous activation of the target gene.
If one is facing the task of activating a human cell gene in a human cell line by gene targeting and obtaining a cell which is capable of the production of the target protein in satisfactory yield and in the desired form, according to the invention one will test several cell lines for the presence of a number of features which this cell line must possess in order to be a suitable candidate for the later large-scale production of the target protein. Preferably, immortalized cell lines, especially tumor cell lines, are tested since they have important advantages over non-immortalized cells regarding culturability.
According to feature (i) the human cell line is studied to see whether the target gene, i.e., the gene to be activated by endogenous gene activation, really has the desired nucleic acid sequence, generally the nucleic acid sequence of the natural target gene. Tumor cell lines and other cell lines in permanent culture often exhibit a series of mutations in their genome. Therefore it is an important aspect in the selection of a suitable cell line whether the cells have a correct gene for the desired product. The sequencing can be done by culturing the cells in the usual manner and sequencing the target gene. If necessary, the target gene can be amplified by PCR or other amplification methods prior to sequencing.
Another important feature for the selection of a cell line is culturability in suspension. Suspension cells are easier to ferment and the fermentation can be adapted more easily to larger dimensions, e.g., in a large fermenter with a capacity of, for example, 10 liters to 50,000 liters. Therefore the selected cells should be either suspension cells or they should easily adapt to a suspension culture. For this purpose the cells are cultured for 14 days with constant stirring. If the cells show at least five population doublings within this period they are considered suitable for suspension culture. Determining the number of population doublings can be done by periodically determining the cell count, e.g., by cell counting or by measuring the optical density of the cell suspension.
Another important feature in the selection of human cells is culturability in a serum-free medium. Since the purification of proteins from serum-free cell cultures is substantially easier and in serum-free culture there is no danger of contamination with animal pathogens, e.g., viruses, the selected cells should be able to grow in a serum-free culture. So the selected cells should be cultured for 14 days in a density of 1 to 10xc3x97105 cells per ml in culture vessels with a serum-free medium (e.g., RPMI 1640 with ITS by Boehringer Mannheim). If the cells during this culture show at least 5 doublings of population, which can be determined by cell counting, they are considered as suitable for serum-free culture.
Another important feature, and one preferred according to the invention is the generation time (iv). The selected cells in media such as, e.g., DMEM 10% fetal calf serum or RPMI 1640 with 10% fetal calf serum, have a high proliferation, i.e., they should have 10 to 256 population doublings, preferably 64 to 128 population doublings, within a week in culture. For this purpose the cells are seeded in culture dishes in a concentration of 0.1 to 10xc3x97105 cells per ml, preferably 0.5 to 2xc3x97105 cells per ml, and the cell count is made every two to three days by means of a cell chamber with or without trypsinization. Cells which show a sufficiently short generation time are especially suitable for large-scale production of human proteins by endogenous gene activation.
Another preferred feature is the absence of any detectable endogenous expression, i.e., transcription and translation, of the target gene (v). Preferably, for the endogenous gene activation, those cell lines are selected which have substantially no endogenous expression of the target gene. For this purpose the cells can be seeded in a cell density of 0.01 to 2xc3x97106 cells/ml, preferably 0.5 to 1xc3x97106 cells/ml of culture medium. After a predetermined time, e.g., 24 hours, the cell supernatant is removed, the cells are discarded, and the content of the target protein is determined in the cell supernatant by known test methods, e.g, ELISA. In the case of EPO the detection limit is, for example, 10 pg/EPO/ml. Cells seeded at 106 cells/ml, which synthesize less than 10 pg of protein are considered as nonproductive and are especially suitable.
Still another important and preferred feature is the polysomia of the target gene in the cell to be selected (vi). The presence of more than two chromosomal copies of the target gene in the cell significantly increases the yields in the homologous recombination. For the production of EPO whose gene is on chromosome 7, the cells Namalwa (Nadkarni et al., Cancer 23 (1969), 64-79) or HeLa S3 (Puck et al., J. Exp. Med. 103 (1956), 273-284), which have chromosome 7 in triplicate, have proven especially suitable. Additional examples of cell lines which contain chromosome 7 in a great number of copies are the colon adenocarcinoma cell line SW-480 (ATCC CCL-228; Leibovitz et al., Cancer Res. 36 (1976), 4562-4567), the malignantmelanoma cell line SK-MEL-3 (ATCC HTB 69; Fogh and Tremp in: Human Tumor Cells in vitro, pp.115-159, J. Fogh (ed.), Plenum Press, New York 1975), the colon adenocarcinoma cell Colo-320 (ATCC CCL-220; Quinn et al., Cancer Res. 39 (1979), 4914-4924), the melanoma cell line MEL-HO (DSM ACC 62; Holzmann et al., Int. J. Cancer 41 (1988), 542-547) and the kidney carcinoma cell line A-498 (DSM ACC 55; Giard et al., J. Natl. Cancer Inst. 51 (1973), 1417-1423).
The examination of the number of chromosomes in the genome of a cell line can be performed by using DNA probes which are specific for the particular chromosome and/or the locus of the target gene.
Still another preferred feature of a starting cell line used for an endogenous gene activation is a correct glycosylation of the desired target protein (vii). A human cell line is preferably used which synthesizes the target protein with a glycosylation pattern, especially regarding the number of the sialic acid moieties, comparable to that of the naturally occurring target protein. The test for the presence of a correct glycosylation is performed preferably by transiently transfecting the cell being tested with an extrachromosomal vector which contains the desired target gene under the control of a promoter active in the cell. After transient expression of the target gene the cell supernatant and/or the cell extract is analyzed by isoelectric focusing. With EPO for example, the presence of correct glycosylation is very evident. In the case of in vitro experiments, non-glycosylated EPO, recombinant EPO from E. coli cells for example, has an activity comparable to that of glycosylated EPO. But in in-vivo experiments non-glycosylated EPO is considerably less active. To determine whether a starting cell line is capable of the production of EPO with correct glycosylation, a comparison can be made with urinary EPO, but also with recombinant EPO from CHO cells, which is known to have an active glycosylated form in humans, and its glycosylation is largely identical with urinary EPO. The comparison of the glycosylation is performed preferably by isoelectric focusing.
Still another preferred feature for the selection of a human cell line is the freedom of the tested cell line from infectious contamination (vii), e.g., from infectious viral particles or mycoplasmas. The test for the presence of viral contamination can be performed by means of cell culture, in vivo analyses and/or detection of specific viral proteins.
The invention moreover relates to a method for preparing human proteins by endogenous gene activation in a human cell line, which is characterized in that a cell line is used which has the features (i), (ii), and (iii) specified above, and preferably at least one of the features (iv), (v), (vi), and (vii), as described above, in addition.
The method of the invention is used especially for the production of human factors, such as EPO, thrombopoietin (TPO), colony stimulating factors, such as G-CSF or GM-CSF, proteins which influence blood coagulation, such as tPA, interferons such as IFN-xcex1, IFN-xcex2or IFN-xcex3, interleukins such as IL-1 to IL-18, chemokines such as MIP, neurotrophic factors such as NGF or BDNF, proteins which affect bone growth such as IFG-BPs, Hedgehog (IgeI) proteins, tumor growth factors such as TFG-xcex2, growth hormones such as HGH, ACTH, encephalins, endorphins, receptors such as, e.g., interleukin or insulin receptors in soluble and/or membrane-standing forms and other protein-binding proteins. With special preference the process for the preparation of EPO is used.
The endogenous gene activation itself can be performed by known methods, and preferably involves the following steps:
(a) Preparation of human starting cell lines which contain at least one copy of an endogenous target gene with the desired nucleic acid sequence, and which, by examining the selection criteria according to the invention, have been identified as suitable for the expression of the target gene,
(b) Transfecting the cells with a DNA construct comprising:
(i) two flanking DNA sequences which are homologous with regions of the target gene locus in order to permit homologous recombination,
(ii) a positive selection marker gene,
(iii) a negative selection marker gene if necessary,
(iv) an amplification gene if desired, and
(v) a heterologous expression control sequence which is active in the human cell
(c) Culturing the transfected cells in conditions which select for the presence of the positive selection marker gene and in some cases the absence of the negative selection marker gene,
(d) Analyzing the cells selectable according to step,
(e) Identifying the cells producing the desired target protein, and
(f) Amplifying the target gene in the selected cells, if desired.
The DNA construct used for making the cell producing the desired human protein contains two flanking DNA sequences homologous to regions of the target gene locus. The choice of suitable flanking sequences is made, for example, according to the methods described in WO90/11354 and WO91/09955. Preferably the flanking sequences have each a length of at least 150 bp.
Especially preferable are the homologous DNA sequences from the 5xe2x80x2 region of the target gene, e.g., 5xe2x80x2-untranslated sequences, signal sequence coding exons and introns located in this region, e.g., exon 1 and intron 1.
The positive selection marker gene can be any selection marker gene suitable for eucaryotic cells which upon expression leads to a selectable phenotype, e.g., antibiotic resistance, auxotrophy etc. The neomycin phosphotransferase gene is an especially preferred positive selection marker gene.
Optionally, a negative selection marker gene may be present, such as the HSV thymidine kinase gene by whose expression cells are destroyed in the presence of a selection agent. The negative selection marker gene is located outside of the two flanking homologous sequence regions.
If an amplification of the target gene endogenously activated in the human cell is desired, the DNA construct contains an amplification gene. Examples of suitable amplification genes are a dihydrofolate reductase gene, an adenosine deaninase gene, an ornithine decarboxylase gene, etc. An especially preferred amplification gene is the dihydrofolate reductase gene, especially a gene coding for a dehydrofolate reductase-arginine mutant, which has a higher sensitivity for the selective agent (methotrexate) than the wild type gene (Simonsen et al., Proc. Natl. Acad. Sci., USA 80 (1983), 2495).
Furthermore, the DNA construct used for endogenous gene activation contains a heterologous expression control sequence which is active in a human cell. The expression control sequence comprises a promoter and preferably additional sequences which improve expression, e.g., an enhancer. The promoter can be an inducible or constitutive promoter. Preferably, the promoter is a strong viral promoter, e.g., an SV40 or CMV promoter. The CMV promoter/enhancer is especially preferred.
The invention furthermore relates to the use of the human cell lines identified by the method described above, after the activation of an endogenous target gene present in the cell to obtain the polypeptide encoded by the activated target gene, preferably to obtain the polypeptide in a large-scale technical process, using, for example, a large fermenter.
Still another object of the invention is a human cell which contains a copy of an endogenous gene in operable linkage with a heterologous expression control sequence active in the human cell, and is capable, without prior gene amplification, of the production of at least 200 ng of the polypeptide/106 cells/24 h. Preferably, the human cell according to the invention is capable of the production of 200 to 3000 ng of polypeptide/106 cells/24 h, and most preferably capable of the production of 1000 to 3000 ng polypeptide/106 cells/24 h.
Finally, still another object of the present invention is a human cell which is obtainable by gene amplification from the cell named above and contains several copies of an endogenous gene, each in operable linkage with a heterologous expression control sequence active in the human cell, and is capable of the production of at least 1000 ng of the polypeptide/106 cells/24 h coded by the endogenous gene. Especially preferred is the human cell line obtainable by gene amplification, capable of the production of 1000 to 25000 ng/polypeptide/106 cells/24 h. Most preferable is a human cell line capable of the production of 5000 to 25,000 ng polypeptide/106 cells/24 hr.
An example of a cell according to the invention is the EPO-producing clone, xe2x80x9cAladin,xe2x80x9d which was deposited on Jul. 15, 1997, according to the provisions of the Budapest Treaty with the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Mascheroder Weg 1b, 38124 Brunswick, under accession number DSM ACC 2320.